Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle.

ABSTRACT: Through the genetic incorporation of a single phenyl azide group into superfolder GFP (sfGFP) at residue 148 we provide a molecular description of how this highly versatile chemical handle can be used to positively switch protein function in vitro and in vivo via either photochemistry or bioconjugation. Replacement of H148 with p-azido-l-phenylalanine (azF) blue shifts the major excitation peak ∼90 nm by disrupting the H-bond and proton transfer network that defines the chromophore charged state. Bioorthogonal click modification with a simple dibenzylcyclooctyne or UV irradiation shifts the neutral-anionic chromophore equilibrium, switching fluorescence to the optimal ∼490 nm excitation. Click modification also improved quantum yield over both the unmodified and origin

SUBMITTER: Hartley AM 

PROVIDER: S-EPMC5355941 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature